Abstract. Myocardial perfusion imaging (MPI), a versatile tool for clinical diagnosis, plays an important role in noninvasive identification of obstructive CAD. Currently, single-photon emission computed tomography (SPECT) MPI agents include 201Tl or 99mTc-complexes, such as 99mTc-sestamibi and 99mTc-tetrofosmin, for determining myocardial perfusion in patients with suspicion of or known CAD. However, SPECT imaging agents have inherent limitations, including the potential 99mMo/99mTc-generator shortage. Additionally, current SPECT tracers also suffer from shortcomings in pharmacokinetics, myocardial extraction, redistribution of the radiotracer to non-targeted tissues over time, and non-linearity of uptake at elevated blood flow (the ?roll-off? phenomenon). Of note, SPECT MPI does not afford the routine clinical calculation of myocardial blood flow (MBF) in ml/g/min as is possible with positron emission tomography (PET). Thus, PET assessment of MBF or myocardial flow reserve (MFR) enables better identification and characterization of both subclinical and clinically-manifest CAD processes and provides important prognostic and therapeutic information, not achievable with SPECT MPI. Commonly employed PET MPI tracers are: 82RbCl, 13NH3, and H215O. Apart from their strengths and weaknesses, these tracers have limited utility for PET imaging due to short half- lives, and restricted access to imaging facilities located within nearby cyclotrons. To enhance access to PET MPI, few promising 18F-labeled agents such as18F-BnTP, a mitochondrial membrane potential probe, and 18F-BMS-747158,18F- 10, 18F-RP1004, and 18F-MCI27, mitochondrial complex I inhibitors, have been investigated in preclinical models. Among these agents, 18F-BMS-747158 (Flurpiridaz, the mitochondrial complex I targeted tracer) has advanced to phase 3 studies. Importantly, complex I deficiency, which is clinically and genetically heterogeneous, can present with hypertrophic cardiomyopathy that could either be isolated or associated with other comorbid multi-organ diseases. Thus, the lower uptake of a given mitochondrial complex I targeted tracer, such as 18F-Flurpiridaz could also be susceptible to these complications. However, these tracers depend on an 18F-radiopharmaceutical distribution business model, which may not readily apply to all sites within the U.S. or other countries in the world. Therefore, PET tracers demonstrating high myocardial first pass extraction, sustained myocardium retention and rapid liver clearance, in vivo stability, and a sufficiently long half-lives to enable PET myocardium imaging with quantification, while incorporating radionuclides that could potentially be generator-produced (rather than cyclotron produced) on site would facilitate wide access to PET MPI technology. To meet this unmet diagnostic clinical nuclear medicine need, we have developed 68Ga- Galmydar which demonstrates high extraction into the myocardium of mice, rats, and rabbits, while displaying efficient clearance from the blood pool. Additionally, 68Ga-Galmydar is also recognized by MDR1 P-glycoprotein (Pgp) and Breast Cancer Resistance Protein (BCRP, ABCG2) as their efficient transport substrate, and thus it exploits transporter- mediated excretion pathways from the blood and liver, resulting in high heart/blood and heart/liver ratios, 60 minute post injection. High resolution live cell-imaging of rat cardiomyoblasts using Galmydar (moderately fluorescent probe), indicates its localization within the mitochondria following permeation within cells thus indicating excellent correlation with radiotracer uptake data. 68Galmydar microPET/CT imaging of rat and rabbit hearts show high myocardial uptake and high target/background contrast. Following ligation of the left anterior descending coronary artery in rat and rabbits, PET/CT imaging clearly visualized the hypoperfused region of the left ventricle wall. These hypoperfused regions of the myocardium correlated well to the same ex vivo regions by histochemical staining post imaging. 68Ga-Galmydar has also shown sensitivity in monitoring mixed ischemia/infarction caused by transient ligation of LAD in rabbits on PET imaging, also with positive correlation of the ischemic region with ex vivo histology (Figure 10). Importantly, Galmydar does not demonstrate any remarkable clinical pathology (toxicology studies) in critical organs following intravenous administration of a single dose (1000 fold excess of an IV imaging dose) of an unlabeled agent into rats over 14 days. Finally, similarly to 13NH3 (a positive control) 68Ga-Galmydar also demonstrates response to adenosine-induced stress in myocardium of rabbits, while indicating nearly identical MBF measurements, employing kinetic modeling (Figure 11). We have also made substantial progress towards kit formulation methodology of 68Ga-Galmydar (Figure 12), to develop technology for enabling onsite production for widespread PET quantitative MPI and myocardial blood flow assessment in patients with obstructive CAD. To further advance translation of this versatile molecular imaging probe into clinic, the specific aims of this revised translational RO1 project are: 1) Validate chemistry manufacturing controls (CMCs) for 68Ga- Galmydar for FDA eIND filing. 2) Formulation, development, and biochemical validation of a single vial kit for preparation of 68Ga-Galmydar. Pharmacokinetic analysis in mice of the 68Ga-Galmydar, prepared using kit formulation methodology. 3) Perform first-in-human studies using 68Ga-Galmydar: evaluate dosimetry, biodistribution, safety, and imaging characteristics following a single injection at rest (n=8, 4 males; 4 females). 4) Evaluate 68Ga-Galmydar potential for noninvasive detection and characterization of subclinical and clinically-manifest CAD in conjunction with PET and its comparative analysis using 13N-NH3 by rest and stress studies in human subjects (n=20, 10 males and 10 females including 10 controls (5 males and 5 females with normal SPECT). Successful execution of proposed aims would deliver widespread quantitative PET MPI and MBF assessment for interrogating obstructive CAD.